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Selection of Functional Glycoforms in Anti-SARS-CoV-2 Human IgG1 Monoclonal Antibodies by FcγRIIIa Affinity
Barbara Oliviero1, Sunil Kumar2, Daniela Conteianni1
1Department of Translational and Clinical Research, Division of Molecular Medicine, Laboratory of Clinical Immunology, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
Monoclonal antibodies activate immune effector cells through Fc-Fcγ receptor interactions, which are strongly influenced by Fc region glycosylation. In this study, three anti-SARS-CoV-2 IgG1 human monoclonal antibodies (hmAbs1-3) derived from the B-cell clones of vaccinated (hmAbs1-2) and convalescent (hmAb3) individuals were investigated, with hmAb3 showing the strongest neutralization activity. Glycoform analysis revealed that hmAb1 predominantly contained ∼60% combined G1F and G2F glycoforms, while hmAb2 consisted of ∼63% G1F, G2F, and G2FS1. In contrast, hmAb3 displayed the greatest glycan diversity with ∼75% comprising G1F, G2F, G2FS1, and G2S1. FcγRIIIa affinity chromatography separated hmAb glycoforms based on receptor affinity, yielding five distinct peaks. Antibody-dependent cellular cytotoxicity (ADCC) assays showed that hmAb3 exhibited the highest activity. Further evaluation of individual hmAb1 fractions collected from the FcγRIIIa affinity column demonstrated a clear correlation between glycosylation patterns and ADCC activity, highlighting the critical roles of Fc galactosylation and sialylation in modulating the effector function.
Monoclonal antibodies activate immune effector cells through Fc-Fcγ receptor interactions, which are strongly influenced by Fc region glycosylation. In this study, three anti-SARS-CoV-2 IgG1 human monoclonal antibodies (hmAbs1-3) derived from the B-cell clones of vaccinated (hmAbs1-2) and convalescent (hmAb3) individuals were investigated, with hmAb3 showing the strongest neutralization activity. Glycoform analysis revealed that hmAb1 predominantly contained ∼60% combined G1F and G2F glycoforms, while hmAb2 consisted of ∼63% G1F, G2F, and G2FS1. In contrast, hmAb3 displayed the greatest glycan diversity with ∼75% comprising G1F, G2F, G2FS1, and G2S1. FcγRIIIa affinity chromatography separated hmAb glycoforms based on receptor affinity, yielding five distinct peaks. Antibody-dependent cellular cytotoxicity (ADCC) assays showed that hmAb3 exhibited the highest activity. Further evaluation of individual hmAb1 fractions collected from the FcγRIIIa affinity column demonstrated a clear correlation between glycosylation patterns and ADCC activity, highlighting the critical roles of Fc galactosylation and sialylation in modulating the effector function.
