Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein

Abstract

MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.

Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging

“…This was likely due to uptake and clearance via the reticular endothelial system and nonspecific binding by proteins in the liver and intestinal tract that can transchelate or bind free copper. We hypothesized that our N-glycosylated HAC-PD1 radiotracer may undergo pattern recognition and clearance by mannose-binding proteins, which can be found as cell surface receptors on macrophages and dendritic cells in the spleen as well as hepatic endothelial cells in the liver (33). To prevent glycosylation of HAC, we mutated the asparagine residues in the NxS/T motifs found in the HAC-PD1 scaffold to aspartic acid.…”

Antibody tumor penetration: Transport opposed by systemic and antigen-mediated clearance

“…This allows a more ideal situation where the tumor is exposed to a large concentration to drive uptake initially, then the clearing agent rapidly reduces the plasma concentration to lower the exposure of normal tissue. Alternatively, yeast glycosylation has been used to increase clearance rates of an ADEPT antibody-enzyme fusion, thereby increasing tumor selectivity, although at a cost of decreased total tumor uptake [128].…”

Engineering Antibodies for Clinical Applications in Cancer

“…This also made it feasible to generate sufficient fusion protein for clinical trials. P. pastoris potentially adds branched mannose at 3 N-glycosylation sites on CPG2; in practice only 2 of the sites were occupied [26].…”