Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Ravetch Laboratory

Abstract

Antibody signaling is a cardinal feature of successful adaptive immune responses. The magnitude and direction of this signaling is determined by the structure of a given antibody’s fragment crystallizable (Fc) domain, which interfaces with cells of the immune system. Often considered a constant region, the immunoglobulin G (IgG) Fc protein backbone and conserved N-linked glycan combine to introduce structural diversity in IgG molecules that in turn, triggers divergent humoral responses. These N-glycans are variably constructed, ultimately leading to families of highly related, but non-equivalent glycoproteins known as glycoforms. Despite burgeoning interest in understanding the complexities of IgG Fc glycoforms and their functions, there is an evident scarcity of tools available to distinguish and target them. In addition, the highly conserved nature of the glycan and its presence on the B cell receptor (BCR) provokes questions of its possible role in the generation of antibody responses. In the first part of this thesis, I identify a novel class of synthetic nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. Applying this technology to Fc glycoforms defines nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. Solving the structure of a nanobody-Fc complex via x-ray crystallography reveals a unique mode of recognition of IgG glycoforms. By adapting nanobodies to standard biochemical methods, I clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their Fc glycan profile, selectively disrupt IgG-Fcγ receptor (FcγR) binding both in vitro and in vivo, and interrogate BCR glycan structure on living cells. In the second part of this thesis, I develop mouse models and biochemical tools to help define the role of the IgG Fc glycan in developing antibody responses. Preliminary studies show that following immunization, mice lacking the N-glycan acceptor residue, Asn 297, exhibit deficiencies in germinal center (GC) responses. In this model, B cells with an aglycosylated BCR participate less frequently in the GC reaction, bind antigen less avidly, and show signaling deficits downstream of BCR engagement. Development of an aglycosylated mouse IgG1-specific nanobody shows that in allelic competition experiments, B cells with glycosylated BCRs are favored. To aid future studies, an additional mouse model null for endogenous Fc-FcγR binding is generated. Finally, a mouse expressing human IgG1 in the heavy chain locus proves useful for chronic administration of human antibodies.

Comments

A Dissertation Presented to the Faculty of the David Rockefeller Graduate Program in Biosciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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