Figure 5. FcγRIIIa binding by human anti-RBD IgGs with variable Fc fucosylation.

(a) Binding of polyclonal IgGs was determined by biolayer interferometry. The overlay of binding traces for a donor from each group representing varying degree of anti-RBD core afucosylation; low (0–10%), medium (10–20%) and high (>20%) is shown. The kinetic constants were obtained by evaluating the binding at multiple concentrations (3.3 mM followed by 2-fold dilutions) of the analyte as shown (solid circles). The fits are indicated by solid lines. The assay was performed twice and shown are representative traces from one experiment. (b) A strong positive correlation (Pearson correlation coefficient r=0.86) was observed between the apparent dissociation constant (KD,app) of FcγRIIIa (CD16a) and anti-RBD IgG1 core afucosylation in COVID-19 patients (n=13) as determined by biolayer interferometry. The binding of anti-RBD monoclonal antibody, CR3022 with different core afucosylation levels is also shown (red). The mean data and the standard error of the mean (SEM) have been graphed. (c) Correlation between the level of anti-RBD IgG1 Fc afucosylation and binding to FcγRIIIa. Binding of serum antibodies (n=38) to FcγRIIIa correlated positively with the degree of afucosylation (Pearson correlation coefficient r=0.64). Samples were representative of the range of Fc fucosylation over the sample set. (d) Correlation between the level of anti-RBD IgG1 Fc afucosylation and immune complex (IC) mediated NK cell degranulation. The amount of degranulation measured by fold increase of CD107a+ NK cells over control correlated positively with the degree of afucosylation of the anti-RBD IgG (Spearman correlation coefficient r= 0.79). The assay was performed in duplicate with PBMCs from three healthy donors and mean data has been graphed. (e, f) Highly afucosylated immune complexes elicited increased production of inflammatory cytokines IL-6, TNF, and IL-1b. Immune complexes were formed using (e) pooled polyclonal IgGs from COVID patients (IL6: p=0.0081 for 100μg/ml, p <0.0001 for 20μg/ml and p=0.0014 for 4μg/ml. TNF: p= 0.0042 for 100μg/ml, p=0.0004 for 20μg/ml and p= 0.0042 for 4μg/ml. IL-1β: p=0.0068 for 100μg/ml, p=0.0065 for 20μg/ml and p=0.0198 for 4μg/ml) or (f) recombinant IgG1 mAb 3022 with distinct levels of afucosylation) (IL6: p=0.0009 for 10μg/ml, p=0.0001 for 2μg/ml and p=0.0017 for 0.4μg/ml. TNF: p= 0.0022 for 10μg/ml, p=0.0008 for 2μg/ml and p= 0.0022 for 0.4μg/ml. IL-1β: p=0.0152 for 10μg/ml, p=0.0231 for 2μg/ml). The assays were performed in duplicate with monocytes from three healthy donors and mean data and standard error of the mean (SEM) has been graphed. P-values between high and low afucosylated immune complexes at each antibody concentration were calculated by two-tailed paired t-tests. *P ≤ 0.05, **P ≤0.01, ***P ≤ 0.001, ****P ≤ 0.0001