Mucin-type fusion proteins with blood group A or B determinants on defined O-glycan core chains produced in glycoengineered Chinese hamster ovary cells and their use as immunoaffinity matrices

Abstract

Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored.