Fcgamma receptors are the primary mediators of IgG effector responses, and individual Fc receptors (FcR) have different affinities for different IgG subclasses1. Four FcgammaRs are present in mice2, and FcgammaRIV (FcgammaRL3, CD16.2) binds to IgG2a, IgG2b3, and IgE4, but not IgG1 or IgG33. FcgammaRIV is a high-affinity receptor for monomeric IgG2a and IgG2b and a low-affinity IgE receptor for both IgEa and IgEb, binding to aggregates but not monomers4. Additionally, IgE immune complexes can displace IgG2 from FcgammaRIV. Surface expression of FcgammaRIV requires gamma chain coexpression in vitro and in vivo3. FcgammaRIV and the gamma chain are upregulated on bone marrow-derived monocytes by IFN-gamma and LPS and are downregulated by TGF-β and IL-4.
According to surface plasmon resonance, 9E9 has strong reactivity to FcgammaRIV as well as low level binding to FcgammaRII and FcgammaRIII2. In vivo, 9E9 binds and blocks FcgammaRIII only when 9E9 first binds FcgammaRIV on the same effector cell, resulting in concurrent inhibition of FcgammaRIII and FcgammaRIV. Native 9E9 binds to FcgammaRII and FcgammaRIII via the Fc.
9E9 was produced by immunizing Armenian hamsters with an FcgammaRIV-IgG1 fusion protein consisting of the extracellular domain of FcgammaRIV fused to a mouse IgG1 Fc portion (D265A-variant deficient in Fc-receptor binding)3. Splenic B cells were then fused to a mouse fusion partner, and hybridoma clones were screened for binding to CHO-K1-FcgammaRIV cells expressing FcgammaRIV.
Blocking studies with 9E9 show that FcgammaRIV is necessary for IgG2a and IgG2b mediated platelet clearance in vivo1. Additionally, blocking FcgammaRIV with 9E9 reduces B-cell depletion2. 9E9 also interferes with immune complex binding to FcgammaRIV3 and can block FcgammaRIII on macrophages and neutrophils2.
