In vitro and in vivo glyco-engineering of human IgG, and the leukocyte-cell surface with new fucosidasen

01 October 2011 → 30 June 2016
Regional and community funding: IWT/VLAIO
Research disciplines
  • Natural sciences
    • Microbiology not elsewhere classified
  • Medical and health sciences
    • Medical systems biology not elsewhere classified
  • Engineering and technology
    • Medical biotechnology not elsewhere classified
fucosidasen microbiology medical biotechnology systems biology
Project description

Glycosylations on proteins are crucial to achieve correct protein folding, and outside the cell glycans perform numerous other functions. A very active area of research currently concerns the analysis of the role of complex sugars in the immune system1 . In particular, we want to explain how these sugars, and specifically fucose, are critical in both the effector function of antibodies and in the migration of leukocytes to specific sites (e.g. 
lymph nodes or an inflamed tissue). Quasi all human IgG molecules are glycosylated with a so-called 'core' fucosylated N-glycan on Asn297 of the heavy chain (CH2 domain). Removal of the core-α-1,6- fucosyl residue causes increased binding between the crystallisable fragment (Fc moiety)
of IgG and its receptor FcγRIIIA (CD16A) by a factor of 302, 3. This leads, compared with fucosylated IgG, to a greatly increased (10-100 ) 'antibody dependent cellular cytotoxicity' (ADCC) activity2, 3. ADCC is the main elimination mechanism for virus-infected cells and tumour cells. This knowledge has led to the development of defucosylated antibodies produced recombinantly in 'Chinese Hamster Ovary' (CHO) cell lines4.

Several of these antibodies are currently undergoing clinical testing. As the 
fucosyltransferase-negative cell lines are not freely available, there is a strong interest in alternative flexible technologies to produce defucosylated antibodies. Moreover, it would be a particularly attractive therapeutic prospect if one were able to defucosylate patient-specific antibodies, thus making them much more potent in their ADCC activity. To date, no technology is available for this. A second immunological role of glycans is in the migration of leukocytes from the blood to inflamed tissues. The first interaction in this extravasation occurs between E- and P-selectin of the activated endothelium and the fucose-containing sialyl-Lewis x (Sia-Lex, CD15s) glycotope5, an Oglycosylation modification on surface glycoproteins of leukocytes. Disruption of this interaction prevents the migration of leukocytes from the blood stream into the inflamed tissue, and is a key target in combating life-threatening acute inflammatory diseases6. 
Drugs that respond to this are under strong investigation, and again, innovative approaches are of great interest.

Glycoside hydrolases (GH), or glycosidases for short, are common enzymes that catalyse the hydrolysis of glycosidic compounds. Their great diversity can be explained by the many functions they perform in nature: from central carbon metabolism and extracting sugars from diet or environment, to protective mechanisms against bacteria and viruses. Clearly, GH, and more specifically fucosidases, could be of particular interest in the above-mentioned therapeutic context. Microorganisms are an eminently rich source of 
secreted glycosidases since polymeric carbohydrates are preferred carbon sources for many micro-organisms. The problem, however, is that in the identification of new glycosidases, it is rarely investigated whether the enzymes are active under physiological conditions on native proteins in human serum or on human blood cells. This leaves the experimental therapeutic approaches just mentioned out of reach so far.