Glycosylation of full-length antibodies in Escherichia coli

DESCRIPTION (provided by applicant): Currently, therapeutic antibodies represent approximately 30% of all biotech revenues (gt 35 billion) and nearly all are produced in mammalian cell culture. Since monoclonal antibodies require glycosylation to provide full therapeutic benefit, mammalian cell culture is the expression platform of choice. Unfortunately mammalian cell culture is slow and expensive with significant product heterogeneity and contamination risk. Although the fastest growing class of therapeutic proteins, monoclonal antibodies can be prohibitively expensive to the health care consumer. Production could be simplified if low-complexity, robust cells were used for large-scale production of fully functional therapeutic antibodies. Full-length monoclonal antibodies (immunoglobulins) can be produced in Escherichia coli that are functionally similar to those produced in mammalian cells except in the ability to bind macrophage Fc receptors and elicit effector function. This inability to elicit effector function is attributed to the lack of glycosylation in the Fc region of E. coli-derived immunoglobulins (IgGs). Recently, it was discovered that the Campylobacter jejuni asparagine-linked (N-linked) protein glycosylation pathway can be functionally transferred into E. coli, conferring the ability to glycosylate proteins. Although the bacterial N-glycan is structurally distinct from its eukaryotic counterparts, this study aims to determine if expression in glycoengineered E. coli is sufficient to rescue binding of full- length IgGs to effector molecules. Of particular interest to this proposal are the C1q receptor of the complement-dependent cytotoxicity pathway and Fc3RI and Fc3RIIIa of the antibody- dependent cellular cytotoxicity pathway. Towards this goal, the objective of this Phase I SBIR proposal is to generate the first full-length IgGs produced in E. coli capable of binding to effectors. To accomplish this we intend to (Aim 1) express and purify N-glycosylated IgGs from glycoengineered E. coli and (Aim 2) determine if glycosylation rescues IgG binding to effector molecules in vitro. These studies are significant because they will (i) explore a paradigm-shifting technology platform for the production of therapeutic proteins and (ii) determine the feasibility of producing glycosylated therapeutic IgGs in E. coli. PUBLIC HEALTH RELEVANCE: The fastest growing class of protein therapeutics, monoclonal antibodies, cannot be produced in Escherichia coli because these simple cells are inherently incapable of performing post- translational glycosylation. Due to the lack of glycosylation in E. coli, these antibodies are not able to elicit effector functions that are often critical for the efficacy of therapeutic antibodies. The focus of these studies is to produce full-length antibodies in glycosylation-competent E. coli and determine if binding to effector molecules is rescued in vitro.