High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells.
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 immunoreactivities 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.
Vailes
Lisa D
LD
INDOOR Biotechnologies Inc, Charlottesville, VA 22903, USA.
Sun
Amanda W
AW
Ichikawa
Kunio
K
Wu
Zining
Z
Sulahian
Timothy H
TH
Chapman
Martin D
MD
Guyre
Paul M
PM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D015202:Protein Engineering; D017452:Receptors, IgG / Q000032:analysis / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus