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Coagulation & its Disorders
N-linked glycosylation modulates
the immunogenicity of recombinant human factor VIII in hemophilia A mice
Ferrata Storti Foundation
Jesse D. Lai,1 Laura L. Swystun,1 Dominique Cartier,1 Kate Nesbitt,1 Cunjie Zhang,2 Christine Hough,1 James W. Dennis2 and David Lillicrap1
1Department of Pathology & Molecular Medicine, Queen’s University, Kingston and 2Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, ON, Canada
ABSTRACT
Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evi- dence 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 con- centrates. 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.
Introduction
The immune response that develops in ~30% of severe hemophilia A (HA) patients remains the most serious complication in factor VIII (FVIII) replacement therapy. Why FVIII-neutralizing antibodies, known as inhibitors, develop in only some patients remains unclear. The type of FVIII concentrate has been proposed as one of the factors that influences the risk of FVIII immunogenicity. Three independ- ent cohort studies have described differences among different recombinant (r) FVIII concentrates, where a 2nd generation full-length rFVIII was associated with a 1.6-fold increase in inhibitor risk compared to 3rd generation products.1-3 Most recently, a fourth retrospective analysis further reported hazard ratios of 2.81 and 1.64 for inhibitor incidence with 2nd and 3rd generation rFVIII, respectively, compared to plas- ma-derived FVIII.4
While these observational studies provide compelling evidence, the mechanistic basis for such findings has only been hypothesized, and not systematically exam- ined.5 The transition from 2nd to 3rd generation rFVIII is based on the removal of human and animal proteins in the production process and final product formulation.
Haematologica 2018 Volume 103(11):1925-1936
Correspondence:
david.lillicrap@queensu.ca
Received: January 11, 2018. Accepted: July 9, 2018. Pre-published: July 12, 2018.
doi:10.3324/haematol.2018.188219
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/11/1925
©2018 Ferrata Storti Foundation
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