Fc Gamma Receptors and Complement Component 3 Facilitate Anti-fVIII Antibody Formation

Abstract

Anti-factor VIII (fVIII) alloantibodies, which can develop in patients with hemophilia A, limit the therapeutic options and increase morbidity and mortality of these patients. However, the factors that influence anti-fVIII antibody development remain incompletely understood. Recent studies suggest that Fc gamma receptors (FcγRs) may facilitate recognition and uptake of fVIII by recently developed or pre-existing naturally occurring anti-fVIII antibodies, providing a mechanism whereby the immune system may recognize fVIII following infusion. However, the role of FcγRs in anti-fVIII antibody formation remains unknown. In order to define the influence of FcγRs on the development of anti-fVIII antibodies, fVIII was injected into WT or FcγR knockout recipients, followed by evaluation of anti-fVIII antibodies. Anti-fVIII antibodies were readily observed following fVIII injection into FcγR knockouts, with similar anti-fVIII antibody levels occurring in FcγR knockouts as detected in WT mice injected in parallel. As antibodies can also fix complement, providing a potential mechanism whereby anti-fVIII antibodies may influence anti-fVIII antibody formation independent of FcγRs, fVIII was also injected into complement component 3 (C3) knockout recipients in parallel. Similar to FcγR knockouts, C3 knockout recipients developed a robust response to fVIII, which was likewise similar to that observed in WT recipients. As FcγRs or C3 may compensate for each other in recipients only deficient in FcγRs or C3 alone, we generated mice deficient in both FcγRs and C3 to test for potential antibody effector redundancy in anti-fVIII antibody formation. Infusion of fVIII into FcγRs and C3 (FcγR × C3) double knockouts likewise induced anti-fVIII antibodies. However, unlike individual knockouts, anti-fVIII antibodies in FcγRs × C3 knockouts were initially lower than WT recipients, although anti-fVIII antibodies increased to WT levels following additional fVIII exposure. In contrast, infusion of RBCs expressing distinct alloantigens into FcγRs, C3 or FcγR × C3 knockout recipients either failed to change anti-RBC levels when compared to WT recipients or actually increased antibody responses, Zerra et al.C3, FcγRs Regulate Anti-fVIII Antibodies depending on the target antigen. Taken together, these results suggest FcγRs and C3 can differentially impact antibody formation following exposure to distinct alloantigens and that FcγRs and C3 work in concert to facilitate early anti-fVIII antibody formation.

Engineering a Therapeutic Protein to Enhance the Study of Anti-Drug Immunity

“…To accomplish this, we first sought to determine whether FVIII-OVA can induce an anti-FVIII antibody response following infusion. To this end, E16 hemophilia A recipients were either exposed to FVIII-OVA or BDD-FVIII, followed by evaluation of anti-FVIII antibody formation, as done previously [ 41 , 80 ]. Exposure to FVIII-OVA induced a robust anti-FVIII antibody response that was very similar to the immune response observed following exposure to BDD-FVIII ( Figure 3 A).…”

“…OTII × Thy 1.1 mice, which are also on a B6 background and possess a TCR specific to OVA 323–339 , were used for splenocyte isolation and adoptive transfer for tracking of FVIII-OVA-specific CD4 T cells [ 64 , 68 ]. HOD mice that express the HOD (hen egg lysozyme fused to OVA and human Duffy) antigen on red blood cells (RBCs) were used as donors [ 60 , 77 , 78 , 79 , 80 , 81 , 82 ]. B6 recipient mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).…”

“…To examine anti-FVIII inhibitor formation in hemophilia A mice following exposure to FVIII, blood was collected from the orbital venous plexus with heparinized capillary tubes 7 days after the last injection of FVIII. A sandwich enzyme-linked immunosorbent assay (ELISA) was used for measuring anti-FVIII IgG and Bethesda assay was used for the measurement of FVIII inhibitor titers, as previously described [ 41 , 76 , 80 , 87 , 88 , 89 ].…”

Complement Inhibition in Severe COVID-19 Acute Respiratory Distress Syndrome

“…Given the patient's worsening multiorgan dysfunction, we reasoned that eculizumab use in this critical circumstance was reasonable given its mechanism of action and mounting evidence in complement-mediated ARDS and AKI, ( 14 – 16 ) and other published reports in COVID-19 ( 6 , 10 – 12 ). After written consent for the off-label use of eculizumab, the patient was given meningococcal and pneumococcal vaccinations and started on a prophylactic antibiotic regimen.…”

Influence of N-glycosylation in the A and C domains on the immunogenicity of factor VIII

“… 37,38 Recent studies 39-41 showed that antibodies may influence de novo antibody formation following exposure to distinct FVIII proteins. FVIII glycosylation may also influence the innate-like B-cell recognition of these glycan epitopes for the initiation of an antibody response, 42 especially when considering that naturally occurring antibodies may facilitate inhibitor formation as was recently shown with xenoglycans. 43 These studies, in addition to the present work, provide an understanding of how FVIII glycosylation may influence antibody formation against FVIII in a variety of contexts.…”