Introduction: Dendritic cells are the predominant antigen presenting cells (APCs) responsible for recognition and processing of pathogens and other proteins to induce a primary immune response. Factor VIII (FVIII) is a highly immunogenic protein and the formation of anti-FVIII antibodies remains the most common complication of hemophilia treatment. The A2 and C2 domains of FVIII account for a significant portion of antibodies formed in patients with severe hemophilia A. Prior studies have shown that C1 domain epitopes mediate FVIII uptake by human and murine monocyte-derived dendritic cells (MDDCs). In contrast, in-vitro uptake of FVIII bound by a human-derived anti-A2 monoclonal antibody (MAb) directed against epitopes Arg484-Ile508 did not alter FVIII intake, suggesting that interactive residues within the A2 domain do not participate in FVIII presentation to APCs. However, in light of the functional and structural similarities between the C1 and C2 domains and current knowledge of the immunogenic B cell epitopes within the C2 domain, further investigation into the C2 domain's role in FVIII uptake by APCs is essential.
Methods:Human peripheral blood mononuclear cells were harvested from healthy donors after written consent. Monocytes were isolated by positive selection using CD14 microbeads. CD14+ monocytes were stimulated and expanded into immature MDDCs in medium containing 1000 U/ml GM-CSF and 800 U/ml IL-4 for 4-6 days. To evaluate FVIII uptake by dendritic cells, MDDCs were incubated with 10 nM DyLight650-conjugated recombinant full length FVIII (DyL650-FVIII) in the absence or presence of a saturating concentration of 80 nM of murine-derived anti-human FVIII MAbs following a 30 minute pre-incubation period of FVIII/MAb for optimal binding at 37°C. MAbs tested were classical anti-C2 MAbs 3D12 and I89, non-classical anti-C2 MAb B9, and anti-A2 MAb 4A4. Cells were subsequently washed, fixed, and analyzed by flow cytometry. Internalization of FVIII and median fluorescence intensity of FVIII uptake by MDDCs in the presence of MAbs were compared to MDDCs exposed to FVIII or serum free medium alone.
Results: Both classical anti-C2 MAbs 3D12 and I89 reduced the percentage of FVIII internalized by MDDCs by 68% and 70% respectively compared to MDDCs treated with FVIII alone similar to the decrease seen with anti-C1 MAbs. Classical anti-C2 MAbs potently inhibit the interaction of FVIII with key binding ligands of Von Willebrand factor (VWF) and phospholipids. The proposed binding epitopes of 3D12, a group B MAb, includes Leu2251, Leu2252, and Phe2196. MAb I89 is a group AB MAb and the proposed binding epitope encompasses residues Met2199 and Phe2200. Non-classical anti-C2 MAb B9 reduced FVIII uptake by 31%, which is comparably less than the classical anti-C2 MAbs. B9 is a group BC antibody that inhibits FVIII binding to phospholipids but does not inhibit binding to VWF. Prior studies have shown that Lys2227 is a critical structural epitope for group BC anti-C2 MAbs. Anti-A2 MAb 4A4, a MAb that binds the Asp403-His444 epitope, did not significantly alter FVIII uptake demonstrating similar internalization of FVIII to the FVIII control. Representative histograms of the percentage of internalized FVIII by untreated MDDCs (shaded gray), MDDCs treated with FVIII alone (black solid line), and MDDCs treated with FVIII in the presence of the anti-C2 or A2 MAbs tested (black dotted line) are shown (Figure 1). Observed reductions in FVIII uptake in the presence of the anti-C2 MAbs suggests that the binding sites recognized by these MAbs play a role in FVIII uptake by dendritic cells.
Conclusion: Binding epitopes recognized by classical and non-classical anti-C2 MAbs mediate FVIII uptake by dendritic cells similar to anti-C1 MAbs. Better understanding of the specific epitopes that promote FVIII uptake and inhibitor formation could ultimately lead to development of mechanisms for inhibitor prediction and prevention.
Meeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.