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

Abstract

A recombinant soluble version of the human high-affinity receptor for IgG, rh-FcRIA 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-FcRIA 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-FcRIA concentration. Aggregated rh-FcRIA lost its ability to inhibit immune complex precipitation and failed to bind to IgG-Sepharose. Addition of human IgG1 to rh-FcRIA prior to incubation at 37°C blocked the formation of rh-FcRIA aggregates. Production of soluble monomeric rh-FcRIA was limited by aggregate formation during cell culture. Substitution of the membrane distal D1 Ig domain of FcRIA with the D1 Ig domain of FcRIIIA or CD16A resulted in a chimeric receptor, FcR3A1A, with enhanced temperature stability. Relative to native rh-FcRIA, FcR3A1A 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-FcRIA and FcR3A1A. These data demonstrate that the D1 Ig domains of FcRIA and FcRIIIA are functionally interchangeable and further suggest that the chimeric receptor FcR3A1A is an effective inhibitor of type III hypersensitivity in mice. © The Author 2010. Published by Oxford University Press. All rights reserved.