The long term objectives of this project are to understand how changes in protein glycosylation regulate antibody dependent immune responses, and then to utilize that knowledge to develop improved antibody-based therapies for diseases such as cancer.
The specific aims of this project are to: 1. Develop methods to produce homogeneous glycoproteins for the study and optimization of antibody glycosylation. 2. Determine the role of N-linked glycosylation in IgG Fc interactions with Fc receptors. 3. Develop methods to incorporate site-specific chemical modifications onto glycosylated IgG Fcs to facilitate biochemical studies and to modify bioactivity. 4. Determine the role of N-linked glycosylation in regulating antibody dependent cellular immune responses. As part of this research, novel methods will be developed to produce homogeneously glycosylated antibody fragments of the immunoglobulin G (IgG) subclasses, and also to attach small molecule receptor ligands to those antibody fragments site-selectively. The glycosylated and chemically modified antibody fragments thus produced will be utilized in in vitro biochemical and structural studies of antibody interactions with Fc receptors to determine the role of glycosylation and IgG subclass in regulating cellular immune responses. Antibody fragments attached to receptor ligands will be utilized to target cancer cells for antibody dependent immune responses in antibody dependent cell-mediated cytotoxicity (ADCC) assays and complement dependent cytotoxicity (CDC) assays. These cell-based assays will be used to optimize antibody dependent immune responses directed against target cells. In the future the knowledge gained by these experiments and the techniques developed will be valuable in developing antibody-based protein therapeutics directed against cancer and other diseases.

Public Health Relevance

Antibodies are important therapeutic proteins that are used to treat cancers, arthritis, and many other diseases. The goal of this project is to understand how modification of human antibodies with sugars regulates antibody dependent immune responses, and then use the knowledge gained from these studies to develop better antibody-based treatments for diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090080-04
Application #
8331610
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Marino, Pamela
Project Start
2010-09-30
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$266,850
Indirect Cost
$78,750
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun et al. (2014) Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297. J Pharm Sci 103:1613-27
Chen, Rui; Pawlicki, Mark A; Tolbert, Thomas J (2014) Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles. Carbohydr Res 383:69-75
Alsenaidy, Mohammad A; Kim, Jae Hyun; Majumdar, Ranajoy et al. (2013) High-throughput biophysical analysis and data visualization of conformational stability of an IgG1 monoclonal antibody after deglycosylation. J Pharm Sci 102:3942-56
Boylan, Nicholas J; Zhou, Wen; Proos, Robert J et al. (2013) Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates. Bioconjug Chem 24:1008-16
Xiao, Junpeng; Tolbert, Thomas J (2013) Modular assembly of dimeric HIV fusion inhibitor peptides with enhanced antiviral potency. Bioorg Med Chem Lett 23:6046-51
Xiao, Junpeng; Tolbert, Thomas J (2011) Site-specific chemical modification of a glycoprotein fragment expressed in yeast. Methods Mol Biol 751:329-42
Chen, Rui; Tolbert, Thomas J (2011) On-resin convergent synthesis of a glycopeptide from HIV gp120 containing a high mannose type N-linked oligosaccharide. Methods Mol Biol 751:343-55