Engineering of therapeutic and diagnostic O-glycans on recombinant mucin-type immunoglobulin fusion proteins expressed in CHO cells.

Metabolic engineering of mammalian cells for optimized glycosylation is usually done to improve activity and the pharmacokinetic features of glycoprotein therapeutics. The field is mainly focused around engineering of N-glycans. We have created a platform in which recombinant mucin-type immunoglobulin fusion proteins are used as scaffolds for multivalent expression of O-glycans with diagnostic or therapeutic potential.

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.

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.

Adsorption of chain type-specific ABO antibodies on Sepharose-linked A and B tetrasaccharides.

Background: Antigen-specific removal of anti-A and anti-B on immunoadsorption columns carrying the blood group A and B trisaccharides is one important component of some protocols used in ABO-incompatible organ transplantation. Because ABO antibodies exist requiring parts of the core saccharide chain for binding, the anti-A and -B-binding capacity of individual and combined, Sepharose-linked Types 1 through 4 A and B tetrasaccharides with that of the A and B trisaccharides was compared.

Study Design And Methods: Sepharose-linked A and B tri- and tetrasaccharides were used to adsorb anti-A and -B from pooled blood group O serum.

Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system.

Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase.

Is there a clinical need for a diagnostic test allowing detection of chain type-specific anti-A and anti-B?

Background: Hemagglutination for detection and semiquantification of ABO antibodies is associated with large center-to-center variations and poor reproducibility. Because acceptance for transplantation and diagnosis of rejection in ABO-incompatible transplantation rely on the levels and specificity of ABO antibodies, reproducible tests that allow their detection and specificity determination are required.

Study Design And Methods: The level of chain type-specific anti-A and anti-B were analyzed in the sera of 44 healthy individuals of known ABO blood group using an enzyme-linked immunosorbent assay (ELISA) with polyacrylamide (PAA) conjugates of blood group A and B trisaccharides or Type 2 chain A and B tetrasaccharides.