Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

Area of Science:

• Immunology
• Hematology
• Protein Engineering

Background:

• Neutralizing anti-drug antibodies (inhibitors) complicate Factor VIII (FVIII) replacement therapy in hemophilia A (HA), affecting 23-35% of severe HA patients.
• Immune tolerance induction (ITI) is the only proven strategy to eradicate inhibitors, but its mechanisms are poorly understood.
• Factor VIII Fc-fusion (rFVIIIFc) shows promise in ITI, possibly due to Fc-mediated interactions beyond half-life extension.

Purpose of the Study:

• To investigate the immunological effects of rFVIIIFc, specifically its interaction with activating Fc gamma receptors (FcγR) and natural killer (NK) cells.
• To elucidate the molecular mechanisms underlying the potential superior efficacy of rFVIIIFc in immune tolerance induction for HA patients.

Main Methods:

• Investigated rFVIIIFc activation of NK cells via FcγRIIIA (CD16) in vitro.
• Assessed rFVIIIFc signaling independent of Von Willebrand Factor (VWF):FVIII complex formation.
• Measured NK cell responses including IFNγ secretion and release of perforin and granzyme B.
• Utilized an antibody-dependent cellular cytotoxicity (ADCC) assay with a B-cell clone expressing an anti-FVIII B-cell receptor.

Main Results:

• rFVIIIFc potently activated NK cells in a CD16-dependent manner, independent of VWF:FVIII complex.
• NK cell activation resulted in significant IFNγ secretion and release of cytolytic molecules (perforin, granzyme B).
• rFVIIIFc-mediated NK cell activation correlated with the high-affinity CD16 (158V) genotype.
• Activated NK cells demonstrated the ability to lyse B-cells expressing anti-FVIII antibodies in an ADCC assay.

Conclusions:

• rFVIIIFc activates NK cells via CD16, leading to cytokine secretion and cytotoxicity, providing a potential mechanism for its enhanced efficacy in ITI for HA patients.
• These findings suggest Fc-fusion proteins could be utilized to target antigen-specific B-cells via NK cell activation for managing adverse immune responses to therapeutic proteins or self-antigens.