Monoclonal antibodies against the C(epsilon)mX domain of human membrane-bound IgE and their potential use for targeting IgE-expressing B cells.
BACKGROUND
IgE mediates immediate-type hypersensitivity reactions responsible for various allergic symptoms. It is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on their surface. The epsilon-chain of human mIgE contains a membrane-anchoring peptide and an extra 52-amino-acid (a.a.)-long domain (referred to as C(epsilon)mX) between the membrane anchor and the CH4 domain.


OBJECTIVE
The study was designed to evaluate the effects of C(epsilon)mX-specific monoclonal antibodies (mAbs) to target IgE-expressing B cells and decrease IgE production.


METHODS
A C(epsilon)mX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting C(epsilon)mX-specific mAbs were obtained.


RESULTS
Characterization of the mAbs using ELISA, immunoprecipitation, and immunoblotting methods showed that they could bind to both native and denatured forms of C(epsilon)mX. The mAbs exhibited mutual inhibition of binding to mIgE. Epitope mapping using synthetic peptides revealed that all five mAbs recognize the same epitope, RADWPGPP, located near the C-terminus of C(epsilon)mX. Binding of one of the mAbs to mIgE on SKO-007 cells induced the cross-linking of mIgE molecules on the cell surface, resulting in their patching and capping. In vitro functional analysis revealed that mAbs are able to cause complement-mediated cytotoxicity on transfectants expressing the Fc portion of mIgE.


CONCLUSIONS
We have prepared several human mIgE-specific mAbs. The potential of the mAbs on targeting mIgE+ B cells was demonstrated by CDC analysis.
Chen
Huan Yuan
HY
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC.
Liu
Fu-Tong
FT
Hou
Charlie M H
CM
Huang
Janice S W
JS
Sharma
Bhavya Bhavna
BB
Chang
Tse Wen
TW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology* / Q000627:therapeutic use; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D003167:Complement Activation; D006224:Cricetinae; D016503:Drug Delivery Systems; D018604:Epitope Mapping; D006825:Hybridomas; D006969:Hypersensitivity, Immediate / Q000628:therapy; D015151:Immunoblotting; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007139:Immunoglobulin epsilon-Chains / Q000737:chemistry / Q000276:immunology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011940:Receptor Aggregation; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007139:Immunoglobulin epsilon-Chains; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins