High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells

Abstract

Background: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. Objective: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. Methods: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22+) or alone (rFel d 1 H22-). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22+ to the CD64 receptor on leukocyte subpopulations and on the THP-1 cell line was analyzed by means of flow cytometry. Results: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22+) and 22 kd (rFel d 1 H22-). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = >0.72, P < .001). The rFel d 1 H22+ bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22+ to THP-1 cells was inhibited by a soluble CD64 fusion protein. Conclusions: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunore-activities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.