Modifications to an Fcgamma-Fcvarepsilon fusion protein alter its effectiveness in the inhibition of FcvarepsilonRI-mediated functions.
BACKGROUND
GE2, a human bifunctional Fcgamma-Fcvarepsilon fusion protein cross-links FcgammaRIIb and FcvarepsilonRI on human mast cells and basophils and results in inhibition of FcvarepsilonRI-mediated functions.


OBJECTIVE
Three modified Fcgamma-Fcvarepsilon (GE) proteins were compared with GE2 for their effect on inhibition of FcvarepsilonRI-mediated cellular responses.


METHODS
GE2 was modified to potentially improve its therapeutic efficacy by increasing binding to FcgammaRIIb (GE S mutant) and decreasing binding to FcgammaRIII (GE H mutant) or reversing the Fcgamma and Fcvarepsilon domains and removing nonhuman linker sequences (E2G). These proteins were tested for their ability to bind a basophil-like cell line, block FcvarepsilonRI-mediated degranulation in human basophils, and inhibit passive cutaneous anaphylaxis in human FcvarepsilonRIalpha-transgenic mice.


RESULTS
All 4 GE proteins bound cells that express FcvarepsilonRI and FcgammaRIIb, although the original GE2 retained the strongest ability to bind to these cells. E2G was as effective as GE2 in its ability to inhibit anti-Fel d 1 IgE-mediated histamine release from human basophils and block passive cutaneous anaphylaxis reactions. The GE S and GE H mutants were less effective.


CONCLUSIONS
Optimization of GE2 as an inhibitor of FcvarepsilonRI-mediated functions showed that effectiveness was maintained when potentially immunogenic linker sequences were removed and Ig domain positions were reversed, but specific residue changes within the IgG C(H)2 domain aimed at enhancing GE2's inhibitory function by increasing FcgammaRII binding or additionally decreasing FcgammaRIII binding were not beneficial.


CONCLUSIONS
GE2 and E2G molecules are effective inhibitors of FcvarepsilonRI-mediated degranulation and are of interest as potential therapeutics for IgE-mediated allergic reactions.
Allen
Lisa Chan
LC
Hart and Louise Lyon Laboratory, Division of Clinical Immunology/Allergy, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Kepley
Christopher L
CL
Saxon
Andrew
A
Zhang
Ke
K
D000818:Animals; D001491:Basophils / Q000378:metabolism / Q000502:physiology; D015550:Cell Degranulation / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D010323:Passive Cutaneous Anaphylaxis / Q000187:drug effects / Q000502:physiology; D020033:Protein Isoforms / Q000494:pharmacology; D017455:Receptors, IgE / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017452:Receptors, IgG / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*
C499173:FCGR2B protein, human; D020033:Protein Isoforms; D017455:Receptors, IgE; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins