High-Performance Glycoengineering of IgE Antibodies

AIMS. To elucidate the role of glycans for IgE biology, and generate optimized IgE glycoforms
REMIT. The project falls within the BBSRC strategic priority areas "New Technologies for the Biosciences" / "Industrial Biotechnology".
BACKGROUND. The global market for biopharmaceuticals is growing at an annual rate of ~14% and is expected to reach $1.6 trillion by 2020. Recombinant therapeutic glycoproteins (RTGs) represent the largest sub-group of these drugs.
The development and manufacture of RTGs remains a considerable biotechnological challenge. RTG glycosylation patterns strongly influence essential properties such as efficacy and safety. However, glycans have high structural complexity, are difficult to analyse, and greatly increase RTG heterogeneity. These factors complicate efforts to understand RTG biology.
We will develop a novel, efficient and cost effective system to study and optimise RTG glycosylation. It will incorporate technologies from three laboratories into workflows for (a) creating diverse RTG glycoforms, (b) studying the structure-activity relationships (SAR) of these, and (c) selecting glycoform populations with optimal properties.
As a proof-of-concept model, the student will use immunoglobulin E (IgE), a key mediator of the allergic immune response in humans. One of the applicants (SK) is leading the clinical development of novel IgE- based therapeutics for cancer immunotherapy. There is evidence that IgE drugs could have efficacy advantages over IgGs. However, IgE antibodies are rapidly cleared in-vivo, with circulatory half-lives of ~1.5-2.5 days, compared to >14 days for IgGs.
Manipulating IgE glycosylation may significantly extend the half-life of IgE drugs. IgE has seven glycosylation sites, yet the effects of IgE glycans on properties such as effector function, stability, half-life and clearance are currently unclear. It is known that terminal galactose residues target glycoproteins to hepatic asialoglycoprotein receptors. This could contribute significantly to the rapid clearance of IgE as within 6-8 hours after dosing IgE shows much higher liver uptake (>50%) than IgG (~5%).

WORK PROGRAMME.
Yr 1: The student will prepare a set of chemical glycosyltransferase inhibitors (e.g. 5-FT-UDP-Gal) and explore different delivery strategies (e.g. ester prodrugs, lipophilic ion-pairs) to maximize cell uptake.
Yr 2: The student will generate novel IgE glycoforms by adding inhibitors to established IgE cultures (e.g. transient HEK293 cells). He/she will analyse their glycan structures with Ludger's state-of-the-art glycan characterisation technologies during an industrial placement.
Yr 3: The student will determine biological properties of IgE glycoforms (e.g. stability, antigen and receptor binding, IgE-mediated signalling and tumour cell killing, apoptosis, proliferation and viability) and carry out additional in-depth glycoanalysis of selected glycoforms during a 2nd placement.
Yr 4: The student will integrate the results from Yrs 2 & 3 to establish IgE glyco-SAR, and identify glycan modifications that confer biotechnological or therapeutic advantages.