N-linked glycosylation modulates the immunogenicity of recombinant human factor VIII in hemophilia A mice

Abstract

Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates. In two hemophilia A mouse models, baby hamster kidney cell-derived factor VIII elicited a stronger immune response compared to Chinese hamster ovary cell-derived factor VIII. Furthermore, factor VIII produced in baby hamster kidney cells exhibited accelerated clearance from circulation independent of von Willebrand factor. Lectin and mass spectrometry analysis of total N-linked glycans revealed differences in high-mannose glycans, sialylation, and the occupancy of glycan sites. Factor VIII desialylation did not influence binding to murine splenocytes or dendritic cells, nor surface co-stimulatory molecule expression. We did, however, observe increased levels of immunoglobulin M specific to baby hamster kidney-derived factor VIII in naïve hemophilia A mice. De-N-glycosylation enhanced immunoglobulin M binding, suggesting that N-glycan occupancy masks epitopes. Elevated levels of immunoglobulin M and immunoglobulin G specific to baby hamster kidney-derived factor VIII were also observed in healthy individuals, and de-N-glycosylation increased immunoglobulin G binding. Collectively, our data suggest that factor VIII produced in baby hamster kidney cells is more immunogenic than that produced in Chinese hamster ovary cells, and that incomplete occupancy of N-linked glycosylation sites leads to the formation of immunoglobulin M- and immunoglobulin G-factor VIII immune complexes that contribute to the enhanced clearance and immunogenicity in these mouse models of hemophilia A.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…As recently reported, rh coagulation factor (rhF) VIII expressed in BHK cells is more immunogenic than that produced in CHO cells (Lai et al, 2018). As recently reported, rh coagulation factor (rhF) VIII expressed in BHK cells is more immunogenic than that produced in CHO cells (Lai et al, 2018).…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…On the other hand, glycans may be protective, acting to shield underlying FVIII epitopes. The presence of glycans shields overlying FVIII protein epitopes and incomplete glycan coverage of the FVIII protein is hypothesised to result in antibody complexes and enhanced immunogenicity (Lai et al , ,b). Although there is a lack of evidence suggesting which hypothesis is more clinically relevant, a recent study proposes that glycans primarily drive the differences in immunogenicity between pdFVIII and rFVIII products even in the presence of its chaperone von Willebrand Factor (VWF) (Jankowski et al , ).…”

“…Recently, it was demonstrated that opsonisation via complement protein C3 plays an important role in enhancing the efficiency of FVIII uptake and the subsequent development of an immune response (Rayes et al , ). Alternatively, antibody‐mediated opsonisation of FVIII via immunoglobulin (Ig)‐M has been observed to increase inhibitor development (Lai et al , ,b), potentially acting through a complement receptor 1‐ or 2‐dependent process (Ferguson et al , ). The binding of IgG to FVIII similarly enhances its uptake but through Fcγ receptor mechanisms (Pincetic et al , ).…”

“…The role of Th17 cells, involved in IL‐17 production and their contribution to autoimmune diseases in general, has recently come to light. T‐cell clones producing IL‐17 have been isolated from HA patients with inhibitors and transcriptomic profiles of the early stages of inhibitor development in mice suggest a pathogenic role for this cytokine that merits further study (Ettinger et al , ; Lai et al , ). IL‐17 production is required for the efficient development of germinal centres (Hsu et al , ).…”

The national blueprint for future basic and translational research to understand factor VIII immunogenicity: NHLBI State of the Science Workshop on factor VIII inhibitors

“…Potential reasons for differences in immunogenicity of FVIII produced by varying cell types need to be better addressed. Interestingly, FVIII produced in cultured cells with different N‐glycosylation showed differences in inhibitor formation in haemophilic mice, albeit no human data have yet been published on this issue . Von Willebrand Factor (vWF), which binds to FVIII and is critical for its half‐life in the circulation, has been shown in several studies to affect FVIII immunogenicity, possibly by changing APC and/or endothelial cell FVIII uptake .…”