Generation of a high-affinity Fcγ receptor by Ig-domain swapping between human CD64A and CD16A

A recombinant soluble version of the human high-affinity receptor for IgG, rh-FcγRIA or CD64A, was expressed in mammalian cells and purified from their conditioned media. As assessed by circular dichroism, size exclusion chromatography and dynamic light scattering, incubation of rh-FcγRIA at 37°C resulted in time-dependent formation of soluble aggregates caused by protein unfolding and loss of native structure. Aggregate formation was irreversible, temperature-dependent and was independent of rh-FcγRIA concentration. Aggregated rh-FcγRIA lost its ability to inhibit immune complex precipitation and failed to bind to IgG-Sepharose. Addition of human IgG1 to rh-FcγRIA prior to incubation at 37°C blocked the formation of rh-FcγRIA aggregates. Production of soluble monomeric rh-FcγRIA was limited by aggregate formation during cell culture. Substitution of the membrane distal D1 Ig domain of FcγRIA with the D1 Ig domain of FcγRIIIA or CD16A resulted in a chimeric receptor, FcγR3A1A, with enhanced temperature stability. Relative to native rh-FcγRIA, FcγR3A1A exhibited less aggregation in Chinese hamster ovary cell-conditioned media or when purified receptor was incubated for up to 24 h at 37°C. Both receptors bound to immobilized human IgG1 with high affinity and were equipotent at blockade of immune complex-mediated cytokine production from cultured mast cells. Equivalent dose-dependent reductions in edema and neutrophil infiltration in the cutaneous Arthus reaction in mice were noted for rh-FcγRIA and FcγR3A1A. These data demonstrate that the D1 Ig domains of FcγRIA and FcγRIIIA are functionally interchangeable and further suggest that the chimeric receptor FcγR3A1A is an effective inhibitor of type III hypersensitivity in mice.