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. 2014 Nov 13;28(17):2523-30.
doi: 10.1097/QAD.0000000000000444.

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function

Amy W Chung  1 , Max Crispin, Laura Pritchard, Hannah Robinson, Miroslaw K Gorny, Xiaojie Yu, Chris Bailey-Kellogg, Margaret E Ackerman, Chris Scanlan, Susan Zolla-Pazner, Galit Alter
Affiliations

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function

Amy W Chung et al. AIDS. .

Abstract

Objective: To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV.

Design: Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions.

Methods: Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC.

Results: Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb.

Conclusions: Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

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Conflict of interest statement

Conflicts of interest

There are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Correlation between specificity and Fc-effector function. (a) The ADCC (% gp140 specific lysis) and (b) ADCP (phagocytic score) activity induced by mAb ordered by increasing strength of responses. (c) Scatter-plot of antibody functional activity by specificity. (d) Gp140SF162 avidity was determined by ELISA. mAb 1418(B19) and 860-55D (CMV) were used as negative controls. 50% maximal binding was calculated for all mAb. Relationship of effector activity and binding avidity was presented as scatter- plots of ADCC (e) and ADCP (f) activity versus gp140SF162 ec50 binding avidity. SPR analysis is shown for (g) FcgRIIa, (h) FcgRIIb, and (i) FcgRIIIa. Relative response to the respective FcgRs was calculated by determining the maximal relative response compared to baseline (time = 0) response during the passing of each mAb sample over each FcgR-coated flow cell between time 25 and 90 s. ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; FcgRs, Fcg receptors; mAb, monoclonal antibody.
Fig. 2
Fig. 2
Correlation between Fc-glycan structures and Fc-function/Fc-receptor binding. (a) Heatmap displaying correlations between Fc-glycan structures and Fc-effector function (ADCP and ADCC)/FcR binding. Nomenclature of glycans: G0, G1, G2, which, respectively, contain 0, 1, 2 galactose residues; single galactose residues are identified as being bound on a-3 or a-6 mannose arm of the core conserved heptasaccharide structure (a-3-arm G1 or a-6-arm G1). Additional residues may include fucose (F), sialic acid (S), bisecting N-acetylglucosamine (B) and N-glycolylneuraminic acid (Neu5Gc). Additionally, galactose can be incorporated in an alpha-1,3 linkage (a-G). Positive correlations are red in color and negative correlations are blue. Significant Spearman rank co-efficient values are identified as *P < 0.05, **P < 0.01. Correlation plots showing: ADCC activity (% gp140-specific lysis) versus (b) total % of G1 glycans bound on the a-3-arm of mannose (a-3-arm-G1), (d) total % of G1 glycans bound on the a-6-arm of mannose (a-6-arm-G1) and (f) total % of fucose minus a-G2F(6)Neu5Gc glycan structures, FcgRIIIa binding versus (c) % total a-3-arm-G1 glycan, (e) % total a-6-arm-G1 glycan and (g) total % of fucose minus a-G2F(6)Neu5Gc glycan structures. ADCC, antibody-dependent cellular cytotoxicity; FcRs, Fc-receptors.
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