Antibody interfaces revealed through structural mining.

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate.

Activation-Induced Cytidine Deaminase Impacts the Primary Antibody Repertoire in Naive Mice.

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire.

Antibody homotypic interactions are encoded by germline light chain complementarity determining region 2.

The utilization of avidity to drive and tune functional responses is fundamental to antibody biology and often underlies the mechanisms of action of monoclonal antibody drugs. There is increasing evidence that antibodies leverage homotypic interactions to enhance avidity, often through weak transient interfaces whereby self-association is coupled with target binding. Here, we comprehensively map the Fab–Fab interfaces of antibodies targeting DR5 and 4-1BB that utilize homotypic interaction to promote receptor activation and demonstrate that both antibodies have similar self-association determinants primarily encoded within a germline light chain complementarity determining region 2 (CDRL2).

Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology.

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions.

Dynamics of heavy chain junctional length biases in antibody repertoires.

Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region of the heavy chain (CDR H3) is the region of highest sequence diversity and is formed by the joining of heavy chain V, D and J germline segments combined with random nucleotide trimming and additions between these segments. We show that CDR H3 and junctional segment length distributions are biased in human antibody repertoires as a function of V, V and J germline segment utilization.

Massively parallel single-cell B-cell receptor sequencing enables rapid discovery of diverse antigen-reactive antibodies.

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion.

Prediction of methionine oxidation risk in monoclonal antibodies using a machine learning method.

Monoclonal antibodies (mAbs) have become a major class of protein therapeutics that target a spectrum of diseases ranging from cancers to infectious diseases. Similar to any protein molecule, mAbs are susceptible to chemical modifications during the manufacturing process, long-term storage, and in vivo circulation that can impair their potency. One such modification is the oxidation of methionine residues.

Barcoded sequencing workflow for high throughput digitization of hybridoma antibody variable domain sequences.

Since the invention of Hybridoma technology by Milstein and Köhler in 1975, its application has greatly advanced the antibody discovery process. The technology enables both functional screening and long-term archival of the immortalized monoclonal antibody producing B cells. Despite the dependable cryopreservation technology for hybridoma cells, practicality of long-term storage has been outpaced by recent progress in robotics and automations, which enables routine identification of thousands of antigen specific hybridoma clones.

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer.

Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER.

Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination.

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response.

Systematic characterization and comparative analysis of the rabbit immunoglobulin repertoire.

Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods.

Differences in the composition of the human antibody repertoire by B cell subsets in the blood.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD(+), memory IgD(-), and plasmacytes isolated from the blood of a single individual.

Identification and characterization of the constituent human serum antibodies elicited by vaccination.

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response.

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells.

Immunoglobulin analysis tool: a novel tool for the analysis of human and mouse heavy and light chain transcripts.

Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization of B cell subpopulations and can shed light on the selective forces that act during immune responses or immune dysregulation, such as autoimmunity, allergy, and B cell malignancy. High-throughput sequencing yields Ig transcript collections of unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable of analyzing large collections of transcripts and provides annotated output files to describe many key properties of Ig transcripts.

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse.

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ(null) engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model.

Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies.