Friday, May 23, 2025
5/23/2025
Engineering mono-fucosylated IgGs to fine-tune antibody-mediated effector functions - Monoclonal IgG antibodies (mAbs) constitute a critically important class of drugs for the treatment of a wide range of diseases. Their abilities to recruit and stimulate immune system cells, which is required for their clinical effectiveness, especially in the immunotherapeutic treatment of cancer, are harbored in their Fc domains. For clinically relevant IgG antibodies, Fc domains engage Fc γ receptors (FcγRs) and complement C1q in order to induce antibody-mediated effector functions that direct the killing of cells in vivo. Methods to engineer IgG Fc domains to manipulate their in vivo killing capacities lag substantially behind those for customizing their antigen- binding Fab domains due to the presence of a conserved N-linked glycan in the Fc domain at residue Asn297 that is overwhelmingly the most important molecular determinant of FcγR and C1q binding. The next generation of immunotherapeutic mAbs depends on our ability to efficiently and rationally modify the chemical structure of this Asn297-linked glycan. The most important molecular feature of this glycan is a fucose sugar unit connected through an α-1,6 linkage to the Asn-proximal N-acetylglucosamine (GlcNac) saccharide. The absence of this core fucose moiety imparts Fc domains with increased binding affinity to FcγR3A, an activating FcγR, resulting in substantially increased antibody-mediated in vivo cellular killing. Naturally produced antibodies, though, are fucosylated and, thus, numerous methods have been developed to create antibodies without fucosylation, resulting in three afucosylated IgG1 antibodies that have recently been approved by the FDA. Antibodies with a fucose on the Asn297-linked glycan on one Fc chain but not on the other – mono-fucosylated antibodies – present a third fucosylation state that could provide fine-tuning of antibody-mediated effector functions, thereby expanding the ability to balance efficacy and toxicity for the treatment of a wide range of diseases. However, mono-fucosylated antibodies do not exist in nature and have never been produced or engineered. Here we propose methods to create mono-fucosylated IgG antibodies for the first time and to assess the biological consequences of antibody mono-fucosylation. We hypothesize that mono-fucosylated IgG antibodies will exhibit biophysical and functional properties distinct from those of fully afucosylated and di-fucosylated IgG antibodies, which can be leveraged to develop a new class of immunotherapeutic monoclonal antibodies that will induce levels of antibody-mediated effector functions optimal for certain clinical indications. This work is significant because it develops and explores an entirely new class of engineered antibodies that could become important for the immunotherapeutic treatment of cancer. The proposed studies are innovative in that they will investigate an entirely novel concept – mono-fucosylated antibodies – with the potential to manipulate antibody-mediated effector functions in ways that have never been explored before. The research plan will be accomplished by leveraging our expertise in IgG-specific glycan remodeling, molecular biophysics and structural biology.
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