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. 2015 Aug 25;112(34):10611-6.
doi: 10.1073/pnas.1513456112. Epub 2015 Aug 7.

A common glycan structure on immunoglobulin G for enhancement of effector functions

Chin-Wei Lin  1 , Ming-Hung Tsai  2 , Shiou-Ting Li  3 , Tsung-I Tsai  3 , Kuo-Ching Chu  2 , Ying-Chih Liu  2 , Meng-Yu Lai  4 , Chia-Yu Wu  3 , Yung-Chieh Tseng  5 , Sachin S Shivatare  2 , Chia-Hung Wang  2 , Ping Chao  2 , Shi-Yun Wang  2 , Hao-Wei Shih  2 , Yi-Fang Zeng  2 , Tsai-Hong You  2 , Jung-Yu Liao  2 , Yu-Chen Tu  2 , Yih-Shyan Lin  2 , Hong-Yang Chuang  2 , Chia-Lin Chen  6 , Charng-Sheng Tsai  2 , Chiu-Chen Huang  2 , Nan-Horng Lin  2 , Che Ma  3 , Chung-Yi Wu  7 , Chi-Huey Wong  8
Affiliations

A common glycan structure on immunoglobulin G for enhancement of effector functions

Chin-Wei Lin et al. Proc Natl Acad Sci U S A. .

Abstract

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

Keywords: Fc glycosylation; endoglycosidase; glycoengineered antibodies; homogeneous antibodies; sugar oxazoline.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) A general strategy for the preparation of homogeneous antibodies with a well-defined glycan structure through in vitro enzymatic remodeling of a mixture of antibody glycoforms. The mixture was first treated with a combination of endoS and the fucosidase from Bacteroides fragilis to generate mono-GlcNAc antibody, followed by ligation with a synthetic glycan oxazoline catalyzed by an endoS mutant. (B) The glycan structures on the homogeneous antibody prepared for the study. G9 can be further extended by glycosyltransferases to form the bisecting 2,3-NSCT-antibody and 2,6-NSCT-antibody.
Fig. 2.
Fig. 2.
Antibody-dependent B-cell depletion of various Rituximab glycoforms. The depletion of human B cells was conducted using freshly prepared human PBMC cells and analyzed with FACS, based on the CD19+ CD3− B cells. (A) Compared with a series of different Rituximab glycoforms, the 2,6-NSCT-Rituximab showed higher depletion activity. (B) In the whole blood B-cell depletion activity of 10 donors, the 2,6-sialylated Rituximab was significantly more active than the nontreated Rituximab, with a P value of 0.0016, whereas the mono-GlcNAc Rituximab showed the lowest activity. (C) The Rituximab-resistant cells of Ramos (Ramos-R) and Raji (Raji-R) express a lower level of CD20 on the cell surface; MFI, medium fluorescence intensity. (D) Ramos and Ramos-R and (E) Raji and Raji-R: The 2,6-NSCT-Rituximab showed remarkable ADCC efficacy toward both normal and resistant cells, whereas the nontreated antibody dramatically lost its activity toward resistant strains.
Fig. 3.
Fig. 3.
EC50 of homogeneous Herceptin glycoforms in the V158 FcγRIIIa mediated ADCC reporter assay. Experiments were performed under the E/T ratio of 6–1 with SKBR3 target cells and the V158 FcγRIIIa engineered effector Jurkat cells. All data shown in the same graph were experiments done in the same microplate and the same batch of effector cells; bars of 95% confidence interval were plotted. (A) The afucosylated Herceptin G8 and the commercial Herceptin showed a similar ADCC effect, indicating that the defucosylation advantage of anti-FcγRIIIa is lost in the afucosylated Herceptin G8. (B) The bisecting and nonbisecting Herceptin analogs G9 and G4 showed similar EC50 values, indicating that the bisecting GlcNAc effect was not observed in this assay. (C) Compared with the glycoengineered Herceptin G1 with two galactose terminals, no significant EC50 change in the 2,6-sialylated antibody was observed, whereas the apparent EC50 increase was shown in the 2,3-sialylated Herceptin. The curves of fold induction were the results of induced luminescence divided by the induction of no antibody control. (D) Samples with the lowest EC50 in A−C were chosen and compared with the commercial Herceptin. All samples demonstrated better activity in this ADCC reporter bioassay.
Fig. 4.
Fig. 4.
Antiinfluenza antibody FI6 with 2,6-NSCT glycan attached to its Fc Asn297 (FI6m) significantly enhances its ADCC activity and prophylactically protects mice from a lethal dose of H1N1 virus challenge. (A) Cytotoxicity is represented as the percentage of lysed HEK293T cells (target cells) expressed with influenza H1 HA (A/California/07/09) when incubated with PBMCs (effector cells) and various concentrations of antibodies. (B) The ADCC activity is shown as fold increases of bioluminescence from a luciferase reporter assay that gave signals when ADCC NFAT pathway was activated. HA-expressed HEK293T cells (target cells) were incubated with NK cells with the aforementioned luciferase reporter (effector cells) and various amounts of antiinfluenza antibody FI6 and FI6m. Curve fitting was conducted with software GraphPad Prism in 4PL nonlinear regression. (C) Survival of mice was monitored upon lethal dose (10 MLD50) infection of influenza virus A/California/07/09 (H1N1). Two hours before infection, each group of mice (n = 9) was administered 2.5 mg/kg of FI6, FI6m, or PBS intraperitoneally. The FI6 and FI6m groups had significant survival difference (P < 0.01).
Fig. 5.
Fig. 5.
A common and optimal N-linked glycan on the Fc region of a therapeutic antibody for the enhancement of antibody activities against infectious and inflammatory diseases, as well as cancer.
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References

    1. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–497. - PubMed
    1. Kang AS, Barbas CF, Janda KD, Benkovic SJ, Lerner RA. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci USA. 1991;88(10):4363–4366. - PMC - PubMed
    1. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: Filamentous phage displaying antibody variable domains. Nature. 1990;348(6301):552–554. - PubMed
    1. Tiller T, et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods. 2008;329(1-2):112–124. - PMC - PubMed
    1. Shields RL, et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J Biol Chem. 2002;277(30):26733–26740. - PubMed

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