Monoclonal antibodies (MAbs) of the immunoglobulin G (IgG) type are an important class of therapeutic glycoproteins. Compelling evidence has indicated that the fine structures of the glycans at the conserved N-glycosylation site (Asn-297) of the Fc domain are responsible for the distinct effector functions of MAbs, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and activation of apoptosis. In addition, a special sialylated Fc glycoform was identified to be responsible for the antiinflammatory activity of intravenous immunoglobulin (IVIG). However, progress in understanding the functional roles of IgG-Fc glycosylation is hampered by the tremendous structural heterogeneity of Fc domain glycans. In addition, controlling glycosylation of MAbs in expression to a desired homogeneous glycoform is still a challenging task. In this application, we propose to explore a chemoenzymatic method to make a library of homogeneously glycosylated IgG-Fc and selected glycoforms of MAbs. Through performing Fc receptor binding studies, we aim to understand how different glycan structures can fine tune the effector functions of IgG and IgG-Fc protein. We have performed important preliminary studies indicating that it is feasible to use the endoglycosidase-based transglycosylation approach to construct defined, homogeneous glycoforms of human IgG-Fc. Building on this success, we propose to pursue three specific aims.
Aim 1 is to explore a chemoenzymatic method for the construction of various pure glycoforms of IgG-Fc.
Aim 2 is to evaluate the structure-activity realtionships of different Fc domain glycoforms in Fc receptor binding, and to evaluate ADCC activity of selectively glycoengineered monoclonal antibodies.
Aim 3 is to synthesize novel Fc domain glycoforms for evaluating the roles of IgG-Fc glycosylation in anti-inflammatory activity. The knowledge gained from the proposed research will eventually facilitate the development of novel glycoforms of MAbs and IgG-Fc proteins as effective therapeutics.

Public Health Relevance

IgG antibodies are an important class of therapeutic glycoproteins. The Fc domain glycosylation is essential for antibody's effector functions including ADCC and anti-inflammatory activity. The proposed research aims to decipher the functional roles of Fc glycosylation through glycosylation engineering and Fc receptor binding studies, which may lead to the discovery of novel antibody glycoforms with potent therapeutic efficacy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM096973-04
Application #
8635370
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (03))
Program Officer
Marino, Pamela
Project Start
2011-07-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
4
Fiscal Year
2014
Total Cost
$300,000
Indirect Cost
$100,000
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Ahmed, Alysia A; Giddens, John; Pincetic, Andrew et al. (2014) Structural characterization of anti-inflammatory immunoglobulin G Fc proteins. J Mol Biol 426:3166-79
Wang, Lai-Xi; Davis, Benjamin G (2013) Realizing the Promise of Chemical Glycobiology. Chem Sci 4:3381-3394
Wang, Lai-Xi; Lomino, Joseph V (2012) Emerging technologies for making glycan-defined glycoproteins. ACS Chem Biol 7:110-22
Zou, Guozhang; Ochiai, Hirofumi; Huang, Wei et al. (2011) Chemoenzymatic synthesis and Fc? receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to Fc?IIIa receptor. J Am Chem Soc 133:18975-91