Multi-Angle Effector Function Analysis of Human Monoclonal IgG Glycovariants

Area of Science:

• Immunology
• Biochemistry
• Therapeutic Antibody Engineering

Background:

• Therapeutic antibody efficacy depends on antigen recognition and Fc-mediated effector functions.
• Fc receptor (FcR) interactions, particularly with Fc gamma receptors (FcγRs), are critical for antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and serum half-life.
• The Fc-linked glycan moiety is essential for IgG effector functionality, influencing FcγR affinity and binding to activating or inhibitory receptor classes.

Purpose of the Study:

• To systematically analyze the effector functions of monoclonal IgG1 and its eight engineered glycosylation variants.
• To investigate the impact of glycosylation on antibody stability and FcγR interactions, both for free antibodies and when bound to antigen or in immune complexes.
• To elucidate the role of glycosylation in IgG1 interaction with FcRn and the influence of immune complex formation on FcR-mediated functions.

Main Methods:

• Enzymatic engineering of monoclonal IgG1 to create eight distinct glycosylation variants.
• Systematic analysis of effector functions, including FcγR binding assays for single antibodies, antigen-bound antibodies, and immune complexes.
• Assessment of structural properties and antibody stability in relation to glycosylation patterns.

Main Results:

• Deglycosylated antibodies showed impaired binding to most FcγRs, except for FcγRI.
• Galactosylation levels directly influenced IgG1 binding to FcγRII and FcγRIIIa, with hypergalactosylated antibodies showing increased interaction.
• Sialylation of antibodies enhanced binding to FcγRIIa and FcγRIIb, and immune complex formation compensated for reduced FcR binding of target-bound antibodies.

Conclusions:

• Glycosylation patterns significantly impact IgG1 stability and FcγR interaction profiles.
• Specific glycan modifications, such as increased galactosylation and sialylation, can modulate FcγR binding and potentially enhance effector functions.
• Immune complex formation is critical for FcR-mediated effector functions, overcoming reduced affinity of target-bound antibodies.