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Widespread Fab-arm exchange affects all endogenous serum IgG4

ABSTRACT: Antibodies are key molecular elements of the human immune system and account for an increasingly large proportion of therapeutics. Next to the well-studied and explored IgG1, several other classes (e.g. IgA, IgM) and sub-classes (e.g., IgG2, IgG3 and IgG4) exist in humans. In particular IgG4 is worth a closer examination as it has unique natural properties and is regularly used as a scaffold in biologicals. In contrast to IgG1, endogenous IgG4 comes in two co-existing structural isomers, owing to the distinct properties of the hinge disulfide-bond connecting the two heavy chains. In one isomer, this disulfide bridge is opened, allowing the release and exchange of one half of the IgG4 molecule, and consequently the formation of bispecific IgG4. Here, we present a LC-MS-based approach enabling the analysis of IgG4 repertoires with clonal resolution, which we used to monitor endogenous serum IgG4 repertoires from seven healthy donors. Most strikingly, our data reveal the combinatorial explosion in diversity of the serum IgG4 clonal repertoire. This phenomenon is explained by the stochastic behavior of Fab-arm exchange, making virtually each IgG4 molecule in serum bispecific. Although the endogenous IgG4 clonal repertoire is therefore extremely diverse, we demonstrate that this IgG4 repertoire persists over time within an individual healthy donor, at least for more than one year.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Albert J.R. Heck

PROVIDER: MSV000097341 | MassIVE | Mon Mar 17 05:51:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (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. Ability of V(D)J recombination to generate vast primary B cell repertoires results from 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 HTGTS 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 non-productive 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. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].

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Widespread Fab-arm exchange affects all endogenous serum IgG4

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