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J.D. Lai et al.
In the aforementioned studies, 2nd and 3rd generation prod- ucts refer specifically to Kogenate FS© (Bayer) produced in baby hamster kidney cells (BHK; BHK-rFVIII), and Advate© (Shire) produced in Chinese hamster ovary cells (CHO; CHO-rFVIII). In addition, these products differ by a single amino acid (AA) at position 1241 in the FVIII B domain, aspartic acid and glutamic acid, respectively. However, the immunologic relevance of this substitution appears insignificant as AA 1241 is poorly represented in both the major histocompatibility class II-restricted pep- tidome of human monocyte-derived dendritic cells (DCs), and the repertoire of DR15-restricted CD4+ T-cell epi- topes.6,7 Commercial formulations of BHK-rFVIII have very recently been reported to contain a higher presence of protein aggregates, which have previously been shown to be immunogenic.8,9
Another hypothesis proposes that the differential immunogenicity is attributed to post-translational modifi- cation of rFVIII, and specifically to differential glycosyla- tion patterns between BHK- and CHO-rFVIII at the 25 potential N-linked sites.5,10 Glycans have been implicated in FVIII intracellular trafficking and folding, as well as clearance by the asialoglycoprotein receptor and Siglec- 5.11-14 High-mannose glycans have been hypothesized to facilitate the uptake of FVIII via the mannose receptor on DCs and macrophages, however the data have been con- flicting, and the in vivo significance of this interaction is unclear.15,16 Previous non-clinical studies have reported similar, or increased, immunogenicity of BHK-rFVIII com- pared to CHO-rFVIII, as well as a decreased, but statisti- cally insignificant, inhibitory antibody response to degly- cosylated FVIII.17-19 No mechanistic explanation for these differences has been provided.
Here, we used the previously described BHK- and CHO-rFVIII concentrates, Kogenate FS® and Advate®, respectively, and assessed their relative immunogenicities in two complementary murine models of HA. We further characterized the glycosylation profiles of each product, and evaluated their role in the development of the anti- FVIII immune response in these murine models.
Methods
FVIII concentrates
The following human rFVIII concentrates were used: Kogenate FS® (full-length BHK-rFVIII; Bayer, Leverkusen, Germany), Advate® (full-length CHO-rFVIII; Shire, Dublin, Ireland), and Xyntha® (CHO-B-domain deleted (BDD)-rFVIII; Pfizer, New York City, NY, USA). Information on FVIII clearance, antigen/activity assays, deglycosylation, and von Willebrand factor (VWF) binding is available in the Online Supplementary Methods.
Mice
Sex and littermate-matched 8-12 week old C57Bl/6 F8 exon 16 knockout (KO) mice with a human full-length F8 transgene con- taining an R593C point mutation (HA-R593C mice) that is tran- scribed, but for which FVIII protein is undetectable in plasma, were used for preliminary experiments.20 Results were extended using similarly controlled “conventional” C57Bl/6 F8 exon 16 KO mice (HA mice).21 Mice were treated by subcutaneous or tail vein intravenous injection of 6 IU (240 IU/kg; as per manufacturer’s label) of rFVIII biweekly for two weeks. Lipopolysacharride (LPS; 1 mg) was used as an adjuvant with the first FVIII infusion where indicated. HA mice were challenged with 2 IU (80 IU/kg) of rFVIII
using the same regimen. Blood was collected by cardiac puncture in one-tenth volume of 3.2% sodium citrate 28 days after the first administration of FVIII. Mouse experiments were approved by the Queen’s University Animal Care Committee.
Anti-FVIII antibodies and FVIII inhibitor assays
FVIII-specific immunoglobulin G (IgG) titres were quantified by enzyme-linked immunosorbent assay (ELISA) and FVIII inhibitors were measured by a 1-stage FVIII clotting assay using an automated coagulometer (Siemens BCS XP, Berlin, Germany), as previously described.22,23 Where indicated, anti-FVIII IgG was quantified using a standard curve generated using the human anti-FVIII monoclonal antibody, EL14 (provided by Dr. Jan Voorberg, Sanquin Research, Amsterdam, The Netherlands).24 Information on human sample collection is available in the Online Supplementary Methods.
FVIII-specific IgM was assessed by indirect ELISA. rFVIII (1 μg/mL) was adsorbed to Nunc Maxisorp 96-well plates overnight. Samples were diluted 1:20 and incubated for 2 hrs. IgM was detected using horseradish peroxidase (HRP)-conjugated goat anti- mouse or anti-human IgM (Southern Biotech, Birmingham, AL, USA). Bovine serum albumin (BSA)-coated wells were used as controls. Plates were developed for 15 minutes using o-phenylene- diamine (Sigma, St. Louis, MO, USA) and read at 492 nm.
Lectin binding assays
rFVIII products were adsorbed to Maxisorp microtitre plates at 1 mg/mL overnight at 4°C. All products saturated binding at this concentration (Online Supplementary Figure S1). Plates were blocked with 1% BSA in phosphate buffered saline (PBS) + 0.01% Tween-20 for 1 hr, and subsequently incubated with biotinylated lectins (Vector Laboratories, Burlingame, CA, USA) for 30 min. Detection was facilitated using streptavidin-poly-HRP (ThermoFisher Scientific, Waltham, MA, USA) and developed for 5 min. Statistical analysis was performed using the Student t test.
FVIII preparation for mass spectrometry
FVIII samples were desalted on ViVaspin, 50kDa MWCO (Sartorius, Goettingen, Germany) spin columns. 20 mg of protein in 400 ml of 50 mM ammonium bicarbonate was reduced in 10 mM dithiothreitol (DTT) at 60°C for 40 min then alkylated in 25 mM iodoacetamide in darkness for 30 min. The reaction was stopped by the addition of 20 mM DTT in darkness for 40 min. Trypsin at 1:50 ratio was added to the sample and incubated at 37°C overnight.
Glycopeptide analysis by liquid chromatography and tandem mass spectrometry (LC–MS/MS)
Peptides were applied to a nano-HPLC Chip using an Agilent 1200 series microwell-plate autosampler interfaced with a Agilent 6550 Q-TOF MS (Agilent Technologies, Santa Clara, CA, USA). The reverse-phase nano-HPLC Chip (G4240-62002) had a 40 nL enrichment column and a 75 mm x 150 mm separation column packed with 5 mm Zorbax 300SB-C18. The mobile phase was 0.1% formic acid in water (v/v) as solvent A, and 0.1% formic acid in ACN (v/v) as solvent B. The flow rate was 0.3 mL/min with gra- dient schedule; 3% B (0-1 min); 3-40% B (1-90 min); 40-80% B(90- 95 min); 80% B (95-100 min) and 80-3% B (100-105 min).
Mascot search was used to identify proteins and sequence cov- erage. Extracted glycopeptides were identified by Agilent Masshunter Quantitative Analysis software by the presence of hexose and N-acetylhexosamine. Glycan structures were predict- ed for extracted glycopeptides by GlycoMod. Glycan structure by MS/MS and occupancy of consensus N_X_S/T N-glycosylation sites were determined manually.
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haematologica | 2018; 103(11)